The response of metazoan cells to apoptotic death signals depends on the status of various regulatory checkpoints in the cell. Prominent among these is the BCL-2 family of proteins whose members include dominant suppressors of cell death (Ced-9, BCL-2, BCL-XL, BCL-w, A1, and MCL-1) and proapoptotic inducers of cell death (BAX, BAK, and BCL-Xs), as well as proapoptotic inhibitors of BCL-2/BCL-X.sub.L function (BAD, BID) (Yang and Korsmeyer, Blood 88:386-401, 1996). The relationship between these family members is complex and, in the case of the BCL-2 suppressor and BAX inducer, is further complicated by their apparent ability to function autonomously in regulating cell death, while at the same time influencing one another's activities via heterodimeric interactions.
BCL-2 suppressors function upstream of caspase death effectors, such as caspase-3, to inhibit cell death. This inhibition of cell death may be accomplished in several ways, including recruitment and regulation of Ced-4-like molecules and Ced-4-like adaptors that are required for activation of initiator caspases, and recruitment of kinases and phosphatases that may regulate the activity of BCL-2-associated complexes. Moreover, regulation of BCL-2 complexes may influence the formation of ion conducting pores or the channel activities of membranes in which BCL-2 resides.
While BAX may affect all of these BCL-2 mediated events via heterodimeric modulation, BAX is also capable of autonomous pore formation in lipid bilayers. The ability of elevated levels of BAX or BAK to initiate cell death in the absence of any additional signal in vivo correlates with severe intracellular membrane dysfunction that includes redistribution of mitochondrial cytochrome c to the cytosol and induced mitochondria permeability transition.
Most BCL-2 and BAX family proteins contain at their extreme COOH-terminus a single predicted transmembrane segment (TM). In the case of BCL-2, the TM functions as a signal-anchor that targets and inserts the protein in a N.sub.cyto -C.sub.in orientation into the two main membrane locations for this protein, the mitochondrial outer membrane and the endoplasmic reticulum/nuclear envelope. Strikingly, however, the ability of BAX to translocate to membrane sites, including mitochondria, is regulated in certain contexts and depends upon the cell receiving a death signal.